Natural Killer T (NKT) cells are a specialized subset of T cells that recognize and respond to alterations in the lipid composition of dendritic cells, macrophages and other types of antigen presenting cells (APCs). They play a significant role in various disease processes, including cancer, allergy, autoimmunity and infection. NKT cells are typically induced by inflammatory cytokines, and are alerted to inflammation by specialized membrane glycoproteins called CD1d molecules. CD1d molecules bind lipids produced by the APCs and present them on the surface, where they are recognized by specific receptors on the NKT cells. When activated, NKT cells rapidly produce a range of cytokines that amplify the subsequent response made by conventional T cells and can determine the type of T cell response that occurs. The overarching goal of this proposal is to determine the parameters that determine the optimal response of NKT cells. In one aim we hypothesize, based on substantial preliminary data, that the glycan chains attached to the CD1d molecules interact with similar glycans on the NKT cell receptor and inhibit the responses of the NKT cells in the absence of inflammation. We will determine which glycans of the CD1d molecule and the NKT cell receptor interact to mediate this effect, and will also determine if modulation of the interaction plays a rle in the recognition of NKT cells in vivo and in vitro. Lipid association with CD1d molecules occurs in internal compartments of the cell called lysosomes, through which CD1d molecules repeatedly traffic from the cell surface. A second goal of the proposal is to understand this process in detail, specifically how lipid trafficking and degradation and CD1d trafficking are integrated to maximize the efficiency of the process. Cellular regulators of the internalization process called Rab GTPases will be assessed for their ability to generate the optimal compartment for lipid binding. Finally, to broaden the search for mechanisms that regulate lipid association we have completed a genetic screen that has revealed at least two novel genes that are required for the process. Determining the functions of these genes is expected to transform our understanding of the NKT cell response to CD1d interactions by identifying mechanisms that do not depend on preconceptions based on known biochemical and cell biological parameters. Ultimately, modulating the actions of these genes may lead to novel therapeutic approaches to the wide variety of diseases in which NKT cells play a role.